MPP1 Antibody

What is MPP1 and why is it important in cellular research?

MPP1 (MAGUK p55 scaffold protein 1) is a multifunctional protein with significant roles in cellular processes. In humans, the canonical protein is 466 amino acid residues with a mass of 52.3 kDa and localizes to the cell membrane . MPP1 exists in three different isoforms produced by alternative splicing and is ubiquitously expressed across tissues .

As a member of the MAGUK protein family, MPP1 serves as an essential regulator of neutrophil polarity, regulating neutrophil polarization through AKT1 phosphorylation independent of PIK3CG activity . It also interacts with spectrin and actin, contributing to cell membrane structural organization and with ankyrin in maintaining erythrocyte membrane structure .

Additionally, MPP1 is also known as M-phase phosphoprotein 1 (MPP1) or KIF20B, functioning as a plus-end-directed kinesin-related protein with microtubule-binding and bundling properties, and microtubule-stimulated ATPase activity .

What are the common applications for MPP1 antibodies in research?

MPP1 antibodies are versatile research tools with multiple validated applications:

For studying MPP1's dynamic localization during cell division, immunofluorescence has proven particularly valuable, revealing its redistribution from interphase nuclei to cytoplasm during metaphase and concentration at the midbody during cytokinesis .

What species reactivity can be expected with commercially available MPP1 antibodies?

Species reactivity varies by antibody product:

Most commercial antibodies target human MPP1, with fewer options validated for mouse and rat models . When selecting an antibody for non-human studies, carefully verify cross-reactivity claims and consider validating the antibody in your specific experimental system.

How should researchers optimize Western blot protocols for effective MPP1 detection?

For optimal Western blot detection of MPP1:

• Sample preparation: Use RIPA buffer for effective extraction of membrane-associated MPP1. For erythrocyte samples, additional considerations are needed as MPP1 is abundant in these cells .

• Gel selection: 10% SDS-PAGE gels are appropriate for resolving the 52.3 kDa MPP1 protein .

• Loading control: When analyzing MPP1 from whole cell lysates, a 30 μg protein load has been validated for HeLa cells .

• Antibody dilution: Most commercial antibodies work effectively at 1:1000 dilution for Western blot applications (e.g., ab96255) .

• Detection method: Both chemiluminescence and fluorescence-based detection methods are compatible with MPP1 antibodies .

• Blocking agent: 5% non-fat milk in TBST is generally effective, though BSA may be preferable when using phospho-specific antibodies to detect post-translationally modified MPP1.

• Positive controls: HeLa and Jurkat cell lysates are well-validated positive controls for MPP1 detection .

What are the key considerations for immunofluorescence studies of MPP1?

When conducting immunofluorescence studies with MPP1 antibodies:

• Fixation method: Methanol fixation (-20°C for 6 min) has been successfully used for MPP1 detection in microtubule studies . For glutaraldehyde-fixed samples, post-fixation treatment with PBS containing 0.1% NaBH₄ prevents autofluorescence .

• Cell types: HEp2 cells, leptomeningeal pericytes, and transfected HEK293T cells have been successfully used to study MPP1 localization . Note that not all cell lines show the same staining pattern; HEK293T cells show weak non-specific staining unless transfected with full-length KIF20B cDNA .

• Co-staining considerations: When studying MPP1's interaction with microtubules, co-staining with anti-tubulin antibodies (e.g., anti-Glu and Δ2 tubulin antibodies) is informative .

• Developmental timing: MPP1 shows cell cycle-dependent localization patterns. In interphase, it localizes to nuclei; during metaphase, it redistributes throughout the cytoplasm; in telophase/anaphase, it concentrates at the midbody .

• Tissue specificity: When examining tissues, particularly strong staining has been observed in cerebellum, ovary, and testis .

How can researchers validate MPP1 antibody specificity to ensure experimental rigor?

Validation of MPP1 antibody specificity is crucial for experimental reliability:

• Multi-antibody approach: Use antibodies targeting different epitopes of MPP1. For comprehensive validation, consider:

  • Antibodies against the N-terminal peptide (e.g., PEMP1: EVRKVRLIQFEKVTEE)

  • Antibodies against the D5 domain peptide (e.g., EAPSCSPFGKKKKYK)

  • Antibodies against the GUK domain

  • Antibodies against the middle region (aa 301-327)

• Knockout/knockdown controls: Test antibodies in MPP1 knockout models or knockdown cell lines. The p55 null mouse model has been validated with multiple antibodies to confirm complete protein absence .

• Preabsorption testing: Perform preabsorption with the immunizing peptide to confirm specificity.

• Peptide competition assays: These can distinguish between specific and non-specific binding.

• Cross-reactivity assessment: Test for cross-reactivity with other MAGUK family members, particularly when using antibodies against conserved domains.

• Positive controls: Use tissues/cells known to express MPP1 (erythrocytes show abundant expression) .

How are MPP1 antibodies being utilized in neurological disorder research?

MPP1 antibodies have emerging applications in neurological research:

• Autoantibody detection: Anti-KIF20B autoantibodies have been detected in up to 25% of patients with idiopathic ataxia, as well as in patients with neuropathies and autoinflammatory conditions . MPP1 antibodies can serve as reference standards for these autoantibody assays.

• Cranial neuropathy research: Anti-MPP1 autoantibodies have been associated with SLE-related cranial neuropathy, making MPP1 antibodies valuable for studying this connection .

• Tissue distribution studies: The 10C7 monoclonal antibody has revealed remarkable staining of specific cell subsets in the cerebellum, providing insights into MPP1's potential role in cerebellar function and related disorders .

• Neuropsychiatric SLE investigations: Using addressable laser bead immunoassay (ALBIA) with purified recombinant MPP1 protein, researchers have identified associations between anti-MPP1 autoantibodies and neuropsychiatric manifestations in SLE patients .

What methodologies enable the study of MPP1's role in neutrophil function?

To investigate MPP1's role in neutrophil polarity and function:

• Neutrophil isolation and manipulation:

  • Isolate neutrophils from human blood or mouse bone marrow

  • Treat with chemoattractants to induce polarization

  • Use MPP1 antibodies to visualize distribution during polarization

• Knockdown/knockout approaches:

  • Utilize neutrophils from MPP1 knockout mice to assess functional consequences

  • Compare morphology and chemotactic responses to wild-type cells

• Phosphorylation studies:

  • Use phospho-specific antibodies to detect MPP1-regulated AKT1 phosphorylation

  • Investigate the phosphorylation-independent mechanism by which MPP1 regulates AKT1

• Live cell imaging:

  • Track MPP1 localization during neutrophil polarization and migration

  • Correlate with cytoskeletal dynamics

• Interaction studies:

  • Perform co-immunoprecipitation with MPP1 antibodies to identify binding partners in neutrophils

  • Investigate interactions with the actin cytoskeleton and regulatory proteins

How can researchers address challenges in detecting post-translational modifications of MPP1?

Studying MPP1 post-translational modifications presents several challenges:

• Phosphorylation detection:

  • MPP1 is phosphorylated at the G2/M transition of the cell cycle

  • The phospho-epitope bound by MPM2 monoclonal antibody was identified as a phosphothreonine consensus sequence Leu-Thr-Pro-Leu-Lys (LTPLK)

  • Use phosphatase inhibitors (e.g., sodium orthovanadate, β-glycerophosphate) in lysis buffers

  • Consider phospho-specific antibodies when available

  • Utilize Phos-tag SDS-PAGE to enhance separation of phosphorylated forms

• Palmitoylation analysis:

  • MPP1 undergoes palmitoylation as a post-translational modification

  • Use hydroxylamine treatment to cleave thioester bonds of palmitoylated proteins

  • Employ biotin-switch techniques or click chemistry-based approaches for palmitoylation detection

  • Consider using inhibitors of palmitoylation to assess functional consequences

• Cell cycle synchronization:

  • For studying cell-cycle dependent modifications, synchronize cells with nocodazole or thymidine block

  • Confirm synchronization with appropriate markers before analyzing MPP1 modifications

• Multiple modification sites:

  • MPP1 may be modified at multiple sites simultaneously

  • Consider mass spectrometry approaches for comprehensive modification mapping

  • Use combinations of specific antibodies to detect different modifications

What are the recommended approaches for studying MPP1's interactions with binding partners?

To investigate MPP1's protein-protein interactions:

• Immunoprecipitation strategies:

  • For optimal results, use 1 mg of total protein from cell lysates (e.g., Jurkat cells) with 6 μg of antibody per reaction

  • Prepare lysates using RIPA buffer for effective solubilization

  • Consider crosslinking approaches for transient interactions

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins can identify proximal interacting partners

  • These approaches are particularly valuable for identifying membrane-proximal interactions

• Co-localization studies:

  • MPP1 colocalizes with various proteins in different contexts:

    • With WHRN at stereocilium tip during hair cell development

    • With MPP5 in the retina at the outer limiting membrane

    • With WHRN in the retina at multiple locations

    • With NF2 in non-myelin-forming Schwann cells

  • Use super-resolution microscopy for precise co-localization assessment

• Interaction mapping:

  • MPP1 contains multiple domains (PDZ, SH3, GUK, L7B, D5) that mediate specific interactions

  • Use domain-specific antibodies or truncated constructs to map interaction regions

• Functional validation:

  • Confirm the biological relevance of identified interactions through functional assays

  • For example, MPP1's interaction with Pin1 is important for regulating protein conformation during cell cycle progression